METABOLIC ENGINEERING FOR GLYCOSYLATION OF NATURAL PRODUCT IN E. COLI

BÁRBARA A. BERCOVICH; HUGO GRAMAJO; EXEQUIEL PORTA; EDUARDO RODRIGUEZ; GUILLERMO LABADIE

Villa General Belgrano - Córdoba

Simposio; Glycobiology Symposium GLYCOAR2016; 2016

Glycosyltransferases(GT) from polyketide gene clusters determine the glycosylation patterns ofmacrolides, which define its biological activities. Previously, we demonstratedthe substrate flexibility of the GT pair MegDI-MegDVI towards its subtrates, aTDP-sugar and a macrolide. This allowed us to produce a newmegosaminyl-azitromycin derivates with improved antimalarial and antibioticactivity. In order to study the structural contribution for antibacterial andantimalarial activity of megosaminyl-azitromycin, new derivatives were producedthrough modifications in desosamine residue introduced by synthetic chemistry. Thusnew megosaminil-azitromycin analogs will be produced and scaled up bybioconversion experiments in a reactor. The structures of these compounds wereconfirmed by mass spectrometry. Once their structure is validated by RMN, theirbiological activity such as antibacterial and antimalarial will be analyzed inorder to test the effect of structural modifications. With the objective tooptimize the production system of megosaminylated-macrolides, newTDP-megosamine operons were constructed using codon-optimazed megDI/DVI genesand introduced in E. coli. Furtheroptimization was carried out by metabolic engineering of of competitiveendogenous pathways that consume the common glu-1-P intermediate. Doublemutations in pgm and glgA/glgC genes were performed andtested for improved carbon flux distribution towards TDP-megosamine when usinggalactose or glucose as carbon source.